Claims:

2. The hybrid molecule of claim 1, wherein the HDAC inhibitor moiety is
modelled after an HDAC inhibitor selected from the group consisting of
TSA, sodium butyrate (NaB), valproic acid, N-acetyldinaline, and
suberoylanilide hydroxamic acid (SAHA).

3. The hybrid molecule of claim 1, wherein the HDAC inhibitor moiety is
derived from TSA.

4. The hybrid molecule of claim 1, wherein the HDAC inhibitor moiety is
derived from SAHA.

5. The hybrid molecule of claims 3 and 4 or a pharmaceutically acceptable
salt or prodrug thereof, as represented by a structure selected from the
group consisting of: ##STR00044## wherein:R1, R2, R3, and
R4 are independently selected from the group consisting of H, lower
alkyl, and alkylene;R5 is selected from the group consisting of H
and OH;X is selected from the group consisting of O, S NH and CH2;Y
is selected from the group consisting of N and CH;m is an integer ranging
from 0 to 3; andn is an integer ranging from 1 to 3.

6. The hybrid molecule of claim 5, or a pharmaceutically acceptable salt
or prodrug thereof comprising the structure: ##STR00045##

7. The hybrid molecule of claim 5, or a pharmaceutically acceptable salt
or prodrug thereof comprising the structure: ##STR00046##

8. The hybrid molecule of claim 5, or a pharmaceutically acceptable salt
or prodrug thereof comprising the structure: ##STR00047##

9. A method for the treatment of disorders or diseases wherein inhibition
of HDAC and/or vitamin D agonism is beneficial, said method comprising
administering to a subject in need thereof and affective amount of one or
more hybrid molecules of claim 1.

10. A method of treating a patient afflicted with a condition selected
from the group consisting of cancer, inflammation and auto-immune
diseases, comprising administering to the patient a therapeutically
effective amount of one or more hybrid molecules of claim 1.

11. A method of inducing wound healing comprising, administering to a
patient in need thereof a therapeutically effective amount of one or more
hybrid molecules of claim 1.

12. A method of treating bacterial infections in a patient comprising,
administering to the patient a therapeutically effective amount of one or
more hybrid molecules of claim 1.

13. A method of reducing proliferation of/or inducing cell death in
neoplastic cells comprising, contacting said neoplastic cells with one or
more of the hybrid molecules of claim 1.

14. Use of one or more of the hybrid molecules of claim 1 in the
manufacture of a medicament for the treatment of a condition selected
from the group consisting of cancer, inflammation and auto-immune
diseases.

15. Use of one or more of the hybrid molecules of claim 1 in the
manufacture of a medicament for inducing wound healing.

16. Use of one or more of the hybrid molecules of claim 1 in the
manufacture of a medicament for treating bacterial infections.

17. A pharmaceutical composition comprising an effective amount of one or
more of the hybrid molecules of 1 in association with one or more
pharmaceutically acceptable carriers, excipients or diluents.

18. An admixture comprising an effective amount of one or more of the
hybrid molecules of claim 1 in association with one or more
pharmaceutically acceptable carriers, excipients or diluents.

Description:

CROSS REFERENCE TO RELATED APPLICATIONS

[0001]The present application claims the benefit of U.S. Provisional
Application No. 60/800,424 filed May 16, 2006, the entire contents of
which are incorporated by reference.

FIELD OF THE INVENTION

[0002]The present invention relates to a series of new chemical agents
that demonstrate antiproliferative and cytotoxic activity against cancer
cells. More particularly, but not exclusively, the present invention
relates to hybrid molecules capable of mixed vitamin D receptor agonism
and histone deacetylase inhibition. The present invention also relates to
methods of their synthesis.

[0004]The vitamin D receptor (VDR), a member of the nuclear receptor
family of ligand-regulated transcription factors, plays a crucial role in
calcitriol's signaling. Calcitriol-bound VDR heterodimerizes with related
retinoid X receptors and binds to specific DNA sequences called vitamin D
response elements, located in the regulatory regions of target
genes.2

[0005]Calcitriol has been reported as regulating cell differentiation and
cell proliferation, as well as having anti-cancer properties.2,3
However, the calcemic activity of calcitriol has limited its use in the
treatment of cancers due to hypercalcemia typically induced by the
required supraphysiological levels of the compound in these treatments.

[0006]Intensive efforts have been devoted to the development of calcitriol
analogues (e.g. suberoylanilide hydroxamic acid, SAHA, 4) that would
combine therapeutic potential with lowered calcemic activity.4-6
Structural changes and modifications in strategic locations throughout
the calcitriol molecular backbone have led to the development of numerous
analogues, many of which have shown potent antiproliferative and
antidifferentiation activities, along with desired lowered calcemic
activity.4 Several of these analogues have advanced to preclinical
studies for the treatment of diverse human diseases, and some have become
FDA-approved drugs.7

##STR00002##

[0007]Cancer progression has been associated with an acquired resistance
to calcitriol. This resistance however, can be overcome by
co-administration of the histone deacetylase (HDAC) inhibitor
trichostatin A (TSA, 3). HDAC inhibitors (a further example of which is
provided by 5) have emerged as a new and promising class of anticancer
agents, capable of regulating transcription and inhibiting cancer cell
proliferation by induction of cell cycle arrest in either G0/G1 or G2/M,
cell differentiation and/or apoptosis.8,9

##STR00003##

[0008]The present description refers to a number of documents, the content
of which is herein incorporated by reference in their entirety.

[0012]In an embodiment, the present invention relates to hybrid molecules
or pharmaceutically acceptable salts thereof selected from the group
consisting of:

##STR00004##

[0013]wherein:

[0014]R1, R2, R3, and R4 are independently selected
from the group consisting of H, lower alkyl, and alkylene;

[0015]R5 is selected from the group consisting of H and OH;

[0016]X is selected from the group consisting of O, S NH and CH2;

[0017]Y is selected from the group consisting of N and CH;

[0018]m is an integer ranging from 0 to 3; and

[0019]n is an integer ranging from 1 to 3.

[0020]In an embodiment, the present invention relates to a hybrid
molecule, or a pharmaceutically acceptable salt or prodrug thereof,
comprising the structure:

##STR00005##

[0021]In an embodiment, the present invention relates to a hybrid
molecule, or a pharmaceutically acceptable salt or prodrug thereof,
comprising the structure:

##STR00006##

[0022]In an embodiment, the present invention relates to a hybrid
molecule, or a pharmaceutically acceptable salt or prodrug thereof,
comprising the structure:

##STR00007##

[0023]In an embodiment, the present invention relates to a method for the
treatment of disorders or diseases wherein inhibition of HDAC and/or
vitamin D agonism is beneficial, the method comprising administering to a
subject in need thereof and affective amount of one or more hybrid
molecules as disclosed herein.

[0024]In an embodiment, the present invention relates to a method of
treating a patient afflicted with a condition selected from the group
consisting of cancer, inflammation and auto-immune diseases, comprising
administering to the patient a therapeutically effective amount of one or
more of the hybrid molecules as disclosed herein.

[0025]In an embodiment, the present invention relates to a method of wound
healing comprising, administering to a patient in need thereof a
therapeutically effective amount of one or more of the hybrid molecules
as disclosed herein.

[0026]In an embodiment, the present invention relates to a method of
treating bacterial infections in a patient comprising, administering to
the patient a therapeutically effective amount of one or more of the
hybrid molecules as disclosed herein.

[0027]In an embodiment, the present invention relates to a method of
reducing proliferation of/or inducing cell death in neoplastic cells
comprising, contacting the neoplastic cells with one or more of the
hybrid molecules as disclosed herein.

[0028]In an embodiment, the present invention relates to a use of one or
more of the hybrid molecules as disclosed herein in the manufacture of a
medicament for the treatment of a condition selected from the group
consisting of cancer, inflammation and auto-immune diseases.

[0029]In an embodiment, the present invention relates to a use of one or
more of the hybrid molecules as disclosed herein in the manufacture of a
medicament for inducing wound healing.

[0030]In an embodiment, the present invention relates to a use of one or
more of the hybrid molecules as disclosed herein in the manufacture of a
medicament for treating bacterial infections.

[0031]In an embodiment, the present invention relates to a pharmaceutical
composition comprising an effective amount of one or more of the hybrid
molecules as disclosed herein in association with one or more
pharmaceutically acceptable carriers, excipients or diluents.

[0032]In an embodiment, the present invention relates to an admixture
comprising an effective amount of one or more of the hybrid molecules as
disclosed herein in association with one or more pharmaceutically
acceptable carriers, excipients or diluents.

[0033]The foregoing and other objects, advantages and features of the
present invention will become more apparent upon reading of the following
non restrictive description of illustrative embodiments thereof, given by
way of example only with reference to the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

[0034]In the appended drawings:

[0035]FIG. 1 is an illustration of the minimized optimal docking structure
of 1 (A) and 6 (B) bound to the VDR ligand binding domain (VDR-LBD), as
obtained using AutoDoc 3.0.® The receptor is shown as ribbons with
the side chains of the amino acid residues and the ligands displayed as
Corey-Pauling-Koltun sticks. Contact residues are labeled in white, with
predicted hydrogen bonds labeled in green. The two hydroxyl moieties of
the A-ring of 1 are within hydrogen bonding distance of polar residues
found in the VDR-LBD, whereas the 25-OH is located between H305 and H397
(A). Twisting of the dienyl side chain of 6 relative to the position of
the side chain in 1, places the hydroxamate OH within hydrogen bonding
distance of H397 (B). Overlay of 1 and 6 in the VDR is shown in C.

[0036]FIG. 2 is an illustration of the vitamin D receptor agonist activity
of 6 using a reporter gene assay in transiently transfected COS7 cells.
Because 4 modestly enhanced expression from the internal control plasmid
expressing β-galactosidase, the data are shown un-normalized with
data for β-galactosidase expression as an inset.

[0037]FIG. 3 is an illustration of the VDR agonist activity of 1, 6, 36
(A) and 1 and 39 (B) measured by determining induction of expression of
the gene encoding CYP24 by reverse transcription/PCR in human head and
neck squamous carcinoma cell (HNSCC) line SCC4. SCC4 cells were treated
with compound concentrations ranging from 0 (-) to 10-6 molar, as
indicated.

[0038]FIG. 4 is an illustration of the HDAC inhibitory activity of 3 in
the SCC4 cell line. FIG. 4A is an illustration of a western blot of
nuclear and cytoplasmic extracts of SCC4 cells treated with vehicle (-),
1, or 3 alone or in combination, as indicated. The 55 kDa band
corresponds to the molecular weight of tubulin. FIG. 4B illustrates a
western blot probed with an antibody directed against acetylated histone
H4 (AcH4) showing the effects of the treatments described in A on
acetylation of histone H4. FIG. 4C illustrates western blots of the
effects of treatments described in A on levels of total alpha-tubulin
(left) and acetylated alpha-tubulin (right).

[0039]FIG. 5 is a comparison of the capability of 3 and 6 (20 nM and 200
nM) in blocking deacetylation of a substrate that absorbs at 405 nm in
its deacetylated form.

[0040]FIG. 6 is a comparison of the HDAC inhibitory activities of 1, 3, 6,
36 or 39 as assessed by their effects on acetylation of alpha-tubulin. In
A, SCC4 cells were incubated with vehicle or compound 1 (10-6M), 3
(15 nM), 6 (10-6M) and 36 (10-6M), as indicated for 6 h and
protein extracts were probed for total α-tubulin (Tub.) or
acetylated α-tubulin (AcTub.) by Western blotting. Blots for
acetylated alpha-tubulin and total alpha-tubulin are shown. In B, SCC4
cells were incubated with vehicle or compound 3 (15 nM), 6 (10-6M)
or 36 (10-6M) as indicated for 6 h or 24 h and protein extracts were
probed for acetylated α-tubulin (AcTub.) by Western blotting. In C,
SCC4 cells were incubated with vehicle or compounds 3 (15 nM), 36
(10-6 or 10-7M) or 39 (10-6 or 10-7M) as indicated
for 6 h or 24 h and protein extracts were probed for total
α-tubulin (tub.) or acetylated α-tubulin (AcTub.) by Western
blotting.

[0041]FIG. 7 is an illustration of the antiproliferative activities of 1
and 3, individually or in combination, in SCC4 cells (A) or in SCC25
cells (B) at the concentrations indicated.

[0042]FIG. 8 is a comparative illustration of the antiproliferative
activities of 1 and 6 in the SCC4 HNSCC (A) and MDA-MB231 (B) breast
cancer cell lines at the concentrations indicated.

[0043]FIG. 9 is an illustration of the effects of 1, 3, 1+3, 6 and 36
administered at the concentrations indicated on cell viability in two
models. The viability of MCF-7 breast cancer cells was monitored after 24
h of incubation using a trypan blue dye exclusion assay (A). The
induction of annexin V, a marker of apoptotic cell death in SCC4 cells,
was measured by FACS analysis (B); 6 induced substantially higher levels
of annexin V staining than 3, 1 or 1+3 after pretreatment with UV light,
which sensitizes cells to apoptotic cell death.

[0044]FIG. 10 is an illustration of the effects of 1, 3, 1+3 and 6 on the
induction of acidic β-galactosidase activity, a marker of autophagy
(A); FIG. 10 is an illustration of the effects 1, 3, 1+3 and 6 on the
levels of β-galactosidase expression as obtained by measuring the
indigo cleavage product of X-Gal at 620 nm (B).

[0045]FIG. 11 is an illustration of the results obtained by
fluorescence-activated cell sorting (FACS) analysis on the distribution
of SCC4 cells in the cell cycle following treatment with vehicle (-), 1,
or 1 and 3 together at the concentrations indicated. The results show
that combined treatment with 1 and 3 induces accumulation of cells in the
G2/M phase of the cell cycle.

[0046]FIG. 12 is an illustration of the immunocytochemical analysis of the
effects of 1, 3, or 1 and 3 combined on SCC4 cells (A). The formation of
tubulin bridges, corresponding to collapsed mitotic spindles (arrows), as
well as an increased number of cell divisions (asterisk), was seen only
in cells treated with 1 and 3 together. Both phenotypes are
characteristics of mitotic catastrophe. The lower panels (B) show
cytoplasmic bridges between SCC4 cells treated with 1 and 3, consistent
with the formation of the tubulin bridges seen above in A.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

[0047]In order to provide a clear and consistent understanding of the
terms used in the present specification, a number of definitions are
provided below. Moreover, unless defined otherwise, all technical and
scientific terms as used herein have the same meaning as commonly
understood to one of ordinary skill in the art to which this invention
pertains.

[0048]The use of the word "a" or "an" when used in conjunction with the
term "comprising" in the claims and/or the specification may mean "one",
but it is also consistent with the meaning of "one or more", "at least
one", and "one or more than one". Similarly, the word "another" may mean
at least a second or more.

[0049]As used in this specification and claim(s), the words "comprising"
(and any form of comprising, such as "comprise" and "comprises"),
"having" (and any form of having, such as "have" and "has"), "including"
(and any form of including, such as "include" and "includes") or
"containing" (and any form of containing, such as "contain" and
"contains"), are inclusive or open-ended and do not exclude additional,
unrecited elements or process steps.

[0050]The term "about" is used to indicate that a value includes an
inherent variation of error for the device or the method being employed
to determine the value.

[0051]The present description refers to a number of chemical terms and
abbreviations used by those skilled in the art. Nevertheless, definitions
of selected terms are provided for clarity and consistency.

[0053]As used herein, the term "alkyl" can be straight-chain or branched.
This also applies if they carry substituents or occur as substituents on
other residues, for example in alkoxy residues, alkoxycarbonyl residues
or arylalkyl residues. Substituted alkyl residues can be substituted in
any suitable position. Examples of alkyl residues containing from 1 to 18
carbon atoms are methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl,
octyl, nonyl, decyl, undecyl, dodecyl, tetradecyl, hexadecyl and
octadecyl, the n-isomers of all these residues, isopropyl, isobutyl,
isopentyl, neopentyl, isohexyl, isodecyl, 3-methylpentyl,
2,3,4-trimethylhexyl, sec-butyl, tert-butyl, or tert-pentyl. A specific
group of alkyl residues is formed by the residues methyl, ethyl,
n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl and tert-butyl.

[0054]As used herein, the term "lower alkyl" can be straight-chain or
branched. This also applies if they carry substituents or occur as
substituents on other residues, for example in alkoxy residues,
alkoxycarbonyl residues or arylalkyl residues. Substituted alkyl residues
can be substituted in any suitable position. Examples of lower alkyl
residues containing from 1 to 6 carbon atoms are methyl, ethyl, propyl,
isopropyl, butyl, isobutyl, tert-butyl, pentyl, isopentyl, neopentyl, and
hexyl.

[0055]As used herein, the term "alkylene" can be a linear saturated
divalent hydrocarbon radical of one to six carbon atoms or a branched
saturated divalent hydrocarbon radical of three to six carbon atoms.
Examples of alkylene residues are methylene, ethylene,
2,2-dimethylethylene, propylene, 2-methylpropylene, butylene, and
pentylene.

[0056]As used herein the term "alkenyl" can be straight-chain or branched
unsaturated alkyl residues that contain one or more, for example one, two
or three double bonds which can be in any suitable position. Of course,
an unsaturated alkyl residue has to contain at least two carbon atoms.
Examples of unsaturated alkyl residues are alkenyl residues such as
vinyl, 1-propenyl, allyl, butenyl or 3-methyl-2-butenyl.

[0057]As used herein the term "alkynyl" can be straight-chain or branched
unsaturated alkyl residues that contain one or more, for example one, two
or three, triple bonds which can be in any suitable position. Of course,
an unsaturated alkyl residue has to contain at least two carbon atoms.
Examples of unsaturated alkyl residues are alkynyl residues such as
ethynyl, 1-propynyl or propargyl.

[0058]As used herein the term "cycloalkyl" can be monocyclic or
polycyclic, for example monocyclic, bicyclic or tricyclic, i.e., they can
for example be monocycloalkyl residues, bicycloalkyl residues and
tricycloalkyl residues, provided they have a suitable number of carbon
atoms and the parent hydrocarbon systems are stable. A bicyclic or
tricyclic cycloalkyl residue has to contain at least 4 carbon atoms. In
an embodiment, a bicyclic or tricyclic cycloalkyl residue contains at
least 5 carbon atoms. In a further embodiment, a bicyclic or tricyclic
cycloalkyl residue contains at least 6 carbon atoms and up to the number
of carbon atoms specified in the respective definition. Cycloalkyl
residues can be saturated or contain one or more double bonds within the
ring system. In particular they can be saturated or contain one double
bond within the ring system. In unsaturated cycloalkyl residues the
double bonds can be present in any suitable positions. Monocycloalkyl
residues are, for example, cyclopropyl, cyclobutyl, cyclopentyl,
cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, cycloheptenyl,
cyclooctyl, cyclononyl, cyclodecyl, cycloundecyl, cyclododecyl or
cyclotetradecyl, which can also be substituted, for example by
C1-C4 alkyl. Examples of substituted cycloalkyl residues are
4-methylcyclohexyl and 2,3-dimethylcyclopentyl. Examples of parent
structures of bicyclic ring systems are norbornane,
bicyclo[2.2.1]heptane, bicyclo[2.2.2]octane and bicyclo[3.2.1]octane.

[0059]As used herein, the term "aryl" means an aromatic substituent which
is a single ring or multiple rings fused together. When formed of
multiple rings, at least one of the constituent rings is aromatic. In an
embodiment, aryl substituents include phenyl and naphthyl groups.

[0061]A combinatory effect of calcitriol and TSA in prostate and breast
cancer has been previously demonstrated.10 Initial studies have
shown a combinatory effect in low nM concentrations of TSA and calcitriol
on the proliferation of calcitriol-resistant SCC4 HNSCC cells (FIG. 7). A
hybrid molecule combining both vitamin D receptor agonism and HDAC
inhibition properties into a single molecular structure was developed.

[0062]Hybrid molecules have had considerable success in pharmacotherapy
and offer several advantages over the use of the individual compounds
(i.e. the compounds making-up the hybrid molecule) in combination
therapy.11-13 Moreover, analyses of dose/toxicity relationships of
hybrid molecules are simpler than those of combination therapies, and
problems associated with differing pharmacokinetic profiles of individual
components are eliminated.

[0063]The design of the hybrid molecules of the present invention is based
on structure-activity relationship (SAR) and X-ray studies. In an
embodiment of the present invention, the hybrid molecule is based on the
structures of calcitriol and TSA. The crystal structure of calcitriol
bound to the VDR-LBD reveals hydrogen bonding to all three hydroxyl
functionalities (Ser237 and Arg274 for 1-OH; Ser278 and Tyr143 for 3-OH;
and His305 and His397 for 25-OH).14 The remainder of the binding
pocket is filled with hydrophobic residues which contact the triene and
C/D-ring sections, as well as a portion of the side chain. The hydrogen
bonding to the hydroxyl functionalities of the A-ring is critical for
binding, as deletion or alteration of the stereochemistry of the 1- or
3-OH group significantly decreases affinity for the VDR.15 Most
potent analogs of calcitriol have hydroxyl moieties in the vicinity of
C-25, although some variation in their exact location (e.g. in EB1089, 4)
is tolerated. The central C/D-ring is less critical, as it may be
partially or fully excised in favor of a single 5- or 6-membered ring or
a linear chain.16,17 19-Nor and C-20 epi analogs are also well
tolerated by the VDR.15,18

[0064]The crystal structure of TSA (3) bound to an HDAC revealed a
tube-like binding pocket possessing a zinc ion coordinated to two Asp
residues and one His residue at a bottom portion of the tube-like binding
pocket.19 The hydroxamic acid function of TSA forms a bidentate
chelate with the zinc ion. There are two other Asp residues and two other
His residues at the bottom portion of the tube-like binding pocket, the
latter of which form hydrogen bonds with the NH and OH groups of the
hydroxamic acid. The polyene chain of TSA spans the remainder of the
tube-like binding pocket, consisting of hydrophobic residues. The top
portion of the tube-like binding pocket terminates at a surface groove
comprising several hydrophobic residues which come into contact with the
dimethylamino group of TSA.

[0065]Based on SAR studies, the γ-methyl dienylhydroxamic acid unit
is required. However, the ketone and adjacent methyl substituted methyne
may be excised, provided that the dimethylamino group is replaced with a
larger unit such as an arylsulfonamide.20 Thus, the dienyl chain in
TSA seems to function as a tether, linking the zinc binding unit with a
"cap" group which binds on the HDAC surface. Hydrogenation of the dienyl
chain in TSA analogs renders them inactive. However, straight chain
analogs lacking the γ-methyl group (e.g. SAHA, 5) have been found
to be potent HDAC inhibitors.

[0066]A first hybrid molecule (6) comprising the 3 hydroxyl moieties
required for binding to the VDR was designed (Scheme 1). The backbone of
the vitamin D core, including the A and C/D-ring systems were maintained
along with the stereochemical relationships of the various substituents.

##STR00008##

[0067]The typical side chain extending off the D-ring of vitamin D was
replaced by a γ-methyl dienylhydroxamic acid unit characteristic of
TSA. It was hypothesized that the hydroxamic acid terminus of this polar
side chain would allow hydrogen bond formation in the active site of the
VDR, as well as permitting chelation to the zinc ion in the HDAC binding
site.

[0068]Preliminary docking experiments using AutoDoc 3.0® indicated
that 6 should bind to the VDR in an orientation substantially similar to
calcitriol. As can be observed from FIG. 1, the minimized structure
retained the critical hydrogen bonds between the A-ring and the receptor
(Ser237 and Arg274 for 1-OH; Ser278 and Tyr143 for 3-OH). Moreover, the
hydroxamic acid is slightly twisted away from the location of the C-25
hydroxyl of calcitriol, preventing a bifurcated hydrogen bond from
forming. However, there is a strong hydrogen bond between the hydroxamate
OH and His397. The affinity of 6 for the receptor was computed to be
between that of calcitriol and EB1089, both of which are effective
ligands in several cancer cell lines.

[0069]In an embodiment, the present invention relates to pharmaceutical
compositions comprising a pharmaceutically effective amount of one or
more hybrid molecules as defined herein, or pharmaceutically acceptable
salts thereof, in association with one or more pharmaceutically
acceptable carriers, excipients and/or diluents. The term
"pharmaceutically effective amount" is understood as being an amount of
hybrid molecule required upon administration to a mammal in order to
induce vitamin D receptor agonism and HDAC inhibition. Therapeutic
methods comprise the step of treating patients in a pharmaceutically
acceptable manner with one or more hybrid molecules or compositions
comprising one or more hybrid molecules as disclosed herein. Such
compositions may be in the form of tablets, capsules, caplets, powders,
granules, lozenges, suppositories, reconstitutable powders, creams,
lotions, or liquid preparations, such as oral or sterile parenteral
solutions or suspensions.

[0070]The therapeutic agents of the present invention (i.e. hybrid
molecules) may be administered alone or in combination with
pharmaceutically acceptable carriers. The proportion of each carrier is
determined by the solubility and chemical nature of the agent(s), the
route of administration, and standard pharmaceutical practice. In order
to ensure consistency of administration, in an embodiment of the present
invention, the pharmaceutical composition is in the form of a unit dose.
The unit dose presentation forms for oral administration may be tablets
and capsules and may contain conventional excipients. Non-limiting
examples of conventional excipients include binding agents such as
acacia, gelatin, sorbitol, or polyvinylpyrolidone; fillers such as
lactose, sugar, maize-starch, calcium phosphate, sorbitol or glycine;
tabletting lubricants such as magnesium stearate; disintegrants such as
starch, polyvinylpyrrolidone, sodium starch glycolate or microcrystalline
cellulose; or pharmaceutically acceptable wetting agents such as sodium
lauryl sulphate.

[0071]The hybrid molecules of the present invention may be injected
parenterally; this being intramuscularly, intravenously, or
subcutaneously. For parenteral administration, the hybrid molecules may
be used in the form of sterile solutions containing solutes, for example
sufficient saline or glucose to make the solution isotonic.

[0072]The hybrid molecules maybe administered orally in the form of
tablets, capsules, or granules, containing suitable excipients such as
starch, lactose, white sugar and the like. The hybrid molecules may be
administered orally in the form of solutions which may contain coloring
and/or flavoring agents. The hybrid molecules may also be administered
sublingually in the form of tracheas or lozenges in which the active
ingredient(s) is/are mixed with sugar or corn syrups, flavoring agents
and dyes, and then dehydrated sufficiently to make the mixture suitable
for pressing into solid form.

[0073]The solid oral compositions may be prepared by conventional methods
of blending, filling, tabletting, or the like. Repeated blending
operations may be used to distribute the active agent(s) (i.e. hybrid
molecules) throughout those compositions employing large quantities of
fillers. Such operations are, of course, conventional in the art. The
tablets may be coated according to methods well known in normal
pharmaceutical practice, in particular with an enteric coating.

[0074]Oral liquid preparations may be in the form of emulsions, syrups, or
elixirs, or may be presented as a dry product for reconstitution with
water or other suitable vehicle before use. Such liquid preparations may
or may not contain conventional additives. Non limiting examples of
conventional additives include suspending agents such as sorbitol, syrup,
methyl cellulose, gelatin, hydroxyethylcellulose, carboxymethylcellulose,
aluminum stearate gel, or hydrogenated edible fats; emulsifying agents
such as sorbitan monooleate or acaci; non-aqueous vehicles (which may
include edible oils), such as almond oil, fractionated coconut oil, oily
esters selected from the group consisting of glycerine, propylene glycol,
ethylene glycol, and ethyl alcohol; preservatives such as for instance
methyl para-hydroxybenzoate, ethyl para-hydroxybenzoate, n-propyl
parahydroxybenzoate, or n-butyl parahydroxybenzoate or sorbic acid; and,
if desired, conventional flavoring or coloring agents.

[0075]For parenteral administration, fluid unit dosage forms may be
prepared by utilizing one or more hybrid molecules and a sterile vehicle,
and, depending on the concentration employed, the hybrid molecule(s) may
be either suspended or dissolved in the vehicle. Once in solution, the
hybrid molecule(s) may be injected and filter sterilized before filling a
suitable vial or ampoule followed by subsequently sealing the carrier or
storage package. Adjuvants, such as a local anesthetic, a preservative or
a buffering agent, may be dissolved in the vehicle prior to use.
Stability of the pharmaceutical composition may be enhanced by freezing
the composition after filling the vial and removing the water under
vacuum, (e.g., freeze drying). Parenteral suspensions may be prepared in
substantially the same manner, except that the hybrid molecule(s) should
be suspended in the vehicle rather than being dissolved, and, further,
sterilization is not achievable by filtration. The hybrid molecule(s) may
be sterilized, however, by exposing it to ethylene oxide before
suspending it in the sterile vehicle. A surfactant or wetting solution
may be advantageously included in the composition to facilitate uniform
distribution of the hybrid molecule(s).

[0076]Topical administration can be used as the route of administration
when local delivery of one or more hybrid molecules is desired at, or
immediately adjacent to, the point of application of the composition or
formulation comprising one or more hybrid molecules.

[0077]The pharmaceutical compositions of the present invention comprise a
pharmaceutically effective amount of one or more hybrid molecules as
described herein and one or more pharmaceutically acceptable carriers,
excipients and/or diluents. In an embodiment of the present invention,
the pharmaceutical compositions contain from about 0.1% to about 99% by
weight of a hybrid molecule as disclosed herein. In a further embodiment
of the present invention, the pharmaceutical compositions contain from
about 10% to about 60% by weight of a hybrid molecule as disclosed
herein, depending on which method of administration is employed.
Physicians will determine the most-suitable dosage of the present
therapeutic agents (i.e. hybrid molecules). Dosages may vary with the
mode of administration and the particular hybrid molecule chosen. In
addition, the dosage may vary with the particular patient under
treatment. The dosage of the hybrid molecule used in the treatment may
vary, depending on the condition, the weight of the patient, the relative
efficacy of the compound and the judgment of the treating physician.

[0078]Synthesis of Hybrid Molecule 6

[0079]The C/D-ring of the vitamin D core of 6, complete with its
stereochemical information, was conveniently prepared by oxidative
degradation of vitamin D2 (17). Attachment of the A-ring via a
Horner coupling of the A-ring phosphine oxide (16) yielded the desired
core backbone. Elongation of the side chain by a series of olefination
reactions yielded a carbonyldienyl moiety which was subsequently
converted into a dienylhydroxamic acid moiety.

[0081]Thus, 7 was converted to the methyl ester 8 and then selectively
bis-silylated on the less sterically hindered hydroxyls to provide 9 in
73% yield. Selective reaction of the secondary hydroxyl of 9 with
1,1-thiocarbonyldiimidazole (TCDI) provided intermediate 10 in excellent
yield (92%). Radical deoxygenation of 10, using NaH2PO2 as the
hydrogen atom source,21 provided 11 in good yields (84%). The
reduction of ester 11 using NaBH4, followed by oxidative cleavage of
vicinal diol 12 using NaIO4, provided ketone 13 in essentially
quantitative yield.

[0082]Homologation of ketone 13 is difficult due to the ease of
elimination of both β-siloxy substituents. The use of less
nucleophilic reagents, particularly those as used in the phosphorous
based olefination reactions, led to the undesired eliminations and
subsequent aromatization to produce phenol. However, the use of the
(TMS)CH2CO2Et/LDA reagent system under Peterson olefination
conditions, provided the α,β-unsaturated ester 14 in good
yield (71%). Ester 14 was subsequently reduced to the allylic alcohol 15
(92%) using DIBAL-H.

[0083]The allylic phosphine 16 was prepared from allylic alcohol 15 via
the in situ formation of a tosylate, followed by displacement with
LiPPh2.22 Subsequent oxidation with aqueous hydrogen peroxide
afforded the desired phosphine oxide 16 in 75% yield following
recrystallization from methanol.

[0085]The residual acid in CHCl3 was sufficient to catalyze
acetalization of the aldehyde functionality of the in situ generated
keto-aldehyde to provide, after a reductive quench with dimethylsulfide,
intermediate 18 in 84% yield. In the absence of CHCl3, the
keto-aldehyde is isolated following a reductive quench of the ozonolysis.
It is important to note that the acetal formation/reductive quench step
must be carefully monitored by TLC, as epimerization of the C-14
stereocenter readily occurs. Indeed, if a stronger acid is used to
catalyze acetal formation, epi-18 is obtained as the major product of the
reaction. Horner coupling of phosphine oxide 16 with keto-acetal 18
provided the vitamin D backbone 19 in 69% yield. Acetal deprotection of
19 was achieved using a 6:3:1 mixture of CHCl3:H2O:TFA at
0° C. Although the deprotection step is slow at this temperature
(3-4 h), careful monitoring of the reaction is required to avoid
epimerization of the C-20 stereocenter.

[0086]E/Z selectivity in the subsequent olefination reactions extending
the side arm is crucial as separation of the isomers would have been
difficult, if at all possible. Wittig olefination of aldehyde 20 provided
the α,β-unsaturated ester 21 in excellent yield (98%) and with
>95:5 E:Z selectivity (Scheme 4).

##STR00011##

[0087]DIBAL-H reduction of the ester provided allylic alcohol 22 (72%),
which was subsequently oxidized to aldehyde 23 (86%) using Dess-Martin
periodinane in the presence of Et3N. Oxidation in the absence of a
weak base resulted in some deprotection of the A-ring hydroxyls due to
the presence of residual acid in the Dess-Martin reagent. A second Wittig
olefination provided the dienyl ester 24 (95%) with the newly generated
double bond being exclusively of the E-configuration. Dienyl ester 24 was
hydrolyzed to carboxylic acid 25 using LiOH in near quantitative yield.
Acid 25 was transformed in situ to the acid chloride prior to treatment
with O-(tert-butyldimethylsilyl)hydroxylamine to produce the
tri-TBS-protected hydroxamic acid which was immediately deprotected using
HF in acetonitrile. Hybrid molecule 6 was isolated in 41% yield from
ester 24, as a white solid after purification by reverse-phase silica gel
chromatography.

[0091]The preparation of 39, complete with its stereochemical information
is based on Scheme 5 as well as illustrated herein below in Scheme 6.

##STR00014##

[0092]Biological Activity

[0093]Hybrid molecule 6 was tested for calcitriol agonist activity using a
reporter gene assay under standard conditions.25,26 COS7 cells were
transiently co-transfected with a plasmid expression vector for the human
VDR, a plasmid vector expressing bacterial β-galactosidase from a
constitutively active promoter (as an internal control for transfection
efficiency), and a vector containing a luciferase reporter gene, under
control of a previously described synthetic promoter composed of three
high affinity VDREs (Vitamin D Response Elements) placed immediately
upstream of a truncated promoter region from the herpes simplex virus
thymidine kinase gene.25 Cells were left overnight in the presence
of DNA and transfection reagent (lipofectamine). The media were changed
and cells were incubated in fresh media containing either vehicle (DMSO),
1 (100 nM), 3 (15 nM), 1 and 3 combined (100 nM/15 nM) or 6 (100 nM), and
incubated a further 36 h. As illustrated in FIG. 2, 3 alone had no
substantial effect on reporter gene activity. Moreover, the calcitriol
agonist activity of 6 is essentially identical to that of 1. The results
were not normalized for β-galactosidase because TSA modestly affects
expression from the internal control plasmid.

[0094]As illustrated in FIG. 3, hybrid molecules 6 and 36 along with 1
(A), and 39 along with 1 (B), were tested for VDR agonist activity in
SCC4 cells treated for 24 h with either vehicle (-) or a range of
concentrations of 1, 6, 36 or 36 from 10-11 to 10-6M. The VDR
agonist activity was assessed by analyzing induction of expression of the
gene encoding CYP24 by reverse transcription/PCR.

[0095]In preliminary experiments, TSA-inducible protein acetylation was
analyzed in nuclear and cytoplasmic extracts of SCC4 cells treated for 6
h with 1, 3, or 1 and 3 and subsequently probed by Western blotting for
protein acetylation using an anti-acetyllysine antibody. As expected, 3
markedly enhanced the acetylation of low molecular weight nuclear
proteins that most likely corresponded to histones, whereas 1 had no
effect alone or in combination with 3 (FIG. 4A, right panel). Effects of
3 on histone acetylation specifically, were confirmed by probing for
acetylation of histone H4 (FIG. 4B). Similar to the results of FIG. 4A, 3
markedly enhanced histone H4 acetylation, while 1 had little effect on
its own or in the presence of 3, indicating that 1 has little effect on
global histone hyperacetylation. In the cytoplasmic extracts, it was
observed that contrary to 3, 1 modestly but consistently enhanced the
levels of a product of ˜70 kDa (FIG. 4A, left panel). However, it
was not clear whether this corresponded to enhanced expression or
acetylation. More strikingly, 3 markedly enhanced the acetylation of a 55
kDa protein, likely to be tubulin. This was confirmed by probing
cytoplasmic fractions with an antibody that recognized α-tubulin
(FIG. 4C). Calcitriol (1) had no effect on the acetylation of
α-tubulin, and none of the treatments had any substantial effect on
total α-tubulin expression (FIG. 4C).

[0096]Hybrid molecule 6 was tested for HDAC inhibitory activity using a
colorimetric assay as illustrated in FIG. 5. Nuclear extracts of SCC4
cells were incubated with vehicle, 3 or 6 in the presence of substrate
for 60 min at 37° C. Following incubation with developer (1 min.),
the absorbance was measured at 405 nM. The results showed that at
equimolar concentrations (20 nM), 6 was less effective at inhibiting HDAC
activity. However, when a ten-fold excess of 6 was employed, (200 nM)
similar HDAC activity was observed indicating that 6 is about 10-fold
less potent than TSA (3).

[0097]As illustrated in FIG. 6A, hybrid molecules 6 and 36 were also
tested for HDAC inhibitory activity by determining their capacity to
enhance acetylation of tubulin in SCC4 cells. SCC4 cells were incubated
with vehicle or compound as indicated for 6 h and cytoplasmic protein
extracts were probed by Western blotting using a specific antibody for
acetylated α-tubulin. The results show that contrary to 36, 1 μM
of 6 induces marked tubulin acetylation. As illustrated in FIG. 6B, SCC4
cells were treated for 6 or 24 h with 10-6 or 10-7M 6, 15 nM 3,
or 10-6 M 36 and extracts were probed for levels of acetylated
α-tubulin. Tubulin acetylation remains elevated in the presence of
6 after 24 h, whereas 36 had no effect on tubulin acetylation at either
time point. As illustrated in FIG. 6C, SCC4 cells were incubated with
vehicle or compound 3 (15 nM), 36 (10-6 or 10-7M) or 39
(10-6 or 10-7M) for 6 h or 24 h and protein extracts were
probed for total alpha-tubulin (tub.) or acetylated alpha-tubulin
(AcTub.) by Western blotting. Elevated levels of acetylated alpha-tubulin
were observed only in cells treated with 3 when results were normalized
for total alpha-tubulin.

[0098]In preliminary experiments, 3 alone completely inhibited SCC4
proliferation at concentrations of 50 nM, while higher concentrations
resulted in substantial cell death (data not shown). As illustrated in
FIG. 7A, cells were therefore treated with 15 nM 3 and either 1 nM 1 or
100 nM 1. As expected, either 1 nM 1 or 100 nM 1 had modest effects on
SCC4 proliferation. However, the combination of low or high concentration
of 1 with 15 nM 3 produced complete growth arrest. As illustrated in FIG.
7B, the more differentiated cell line SCC25 was relatively more sensitive
to 1 at low or high concentrations and substantially less sensitive to 15
nM 3. Only the combination of 3 with 100 nM 1 produced completely blocked
proliferation of SCC25 cells.

[0099]As illustrated in FIG. 8, hybrid molecule 6 was tested for its
antiproliferative activity in the human cancer cell lines SCC4 and
MDA-MB231. The SCC4 cell line (A) is representative of cells from
advanced, de-differentiated squamous tumors and is resistant to the
antiproliferative effects of 1 and its analogues.23,24 Subconfluent
SCC4 cells were treated with vehicle (DMSO), 1 or 6 over a 96 h period.
Tissue culture media was changed daily and fresh 1, 6 or vehicle were
added. Under these conditions, 6 exhibited greater efficacy than 1 in
inhibition of SCC4 proliferation. Similarly, treatment of the estrogen
receptor negative breast cancer cell line MDA-MB231 (B), derived from a
metastatic breast tumor, demonstrated similar potency and efficacy of 6.
Data obtained in the prostate cancer cell lines PC3 and Du145 were
similar but are not shown.

[0100]The effect of 1, 3, 1+3, 6 and 36 on cell viability was tested in
two models. The viability of MCF-7 breast cancer cells was monitored
after 24 h of incubation using a trypan blue dye exclusion assay (FIG.
9A). The results show that 6 induced markedly more cell death at
concentrations of 10-7 or 10-6M than 1 at 10-6M or the
combination of 1 and 3, whereas the effects of 36 were similar to those
of 1. The effects of 100 nM 1, 15 nM 3 (TSA), 1 and 3 combined (100 nM/15
nM) and 100 nM 6 (hybrid molecule) on cell viability were tested by
screening for induction of markers of apoptotic cell death. Annexin V
staining was screened by FACS analysis as in Tavera-Mendoza et al.26
While all treatments induced modest increases in annexin V staining,
apoptosis could be excluded as the major cause of cell death (FIG. 9B).
However, it was noted that 6 induced substantially higher levels of
annexin V staining than 3, 1 or 1+3 after pretreatment with UV light,
which sensitizes cells to apoptotic cell death.

[0101]Since it was observed that treatment with 1 could enhance cell death
by autophagy (ref. 26), the effects of different treatments on the
induction of acidic β-galactosidase activity, a marker of autophagy
(FIG. 10A) was tested. Cytochemical analysis of β-galactosidase
expression by staining with
5-bromo-4-chloro-3-indoxyl-β-D-galactoside (X-Gal) at pH 4.0 in
control (DMSO)-treated cells revealed none or very little staining.
Treatment with 3 had weak or relatively modest effects on
β-galactosidase levels. Treatment with 1 or 1+3 generally enhanced
β-galactosidase staining, as did treatment with 6. While these are
qualitative analyses, it was noted that treatment with 6 produced a
higher percentage of cells that stained intensely for perinuclear
β-galactosidase expression, characteristic of formation of
autophagosomes. The effects of each treatment on the induction of acidic
β-galactosidase expression was therefore assessed more
quantitatively by spectrophotometric measurement of the indigo cleavage
product of X-Gal at 620 nm.27 These studies revealed that treatment
with 6 induced ˜30% higher levels of β-galactosidase
expression than treatment with 1 or 1+3 (FIG. 10B).

[0102]Taken together, the results illustrated in FIGS. 9 and 10 provide
evidence that 6 displays enhanced biochemical activity over the
combination of 1 and 3. As shown in FIG. 9, hybrid molecule 6 induced
substantially higher levels of apoptosis as measured by annexin V
staining than 1 and 3 in SCC4 squamous carcinoma cells sensitized for
apoptotic cell death. As illustrated in FIG. 10, both cytochemical and
quantitative analysis indicated that cells treated with 6 displayed
elevated levels of autophagy in SCC4 cells, which also leads to cell
death. These results suggest that hybrid molecules such as 6, which
combine vitamin D agonism with HDAC inhibition, have enhanced therapeutic
activity over combinations of vitamin D agonists and HDAC inhibitors.

[0103]The combined effects of 1 and 3 on the distribution of SCC4 cells in
the cell cycle were assessed by FACS analysis (FIG. 11). Cells were
treated with vehicle, 100 nM 1, or a combination of 100 nM 1 and 15 nM 3
for 72 h, as indicated. Calcitriol (1) alone augmented the percentage of
cells in either G1 or G2/M and substantially diminished the proportion of
cells in S phase. While the combination of 1 and 3 diminished the
proportion of cells in S phase, as expected, it also diminished the
proportion of cells in G1 and markedly enhanced the number of cells in
G2/M, indicative of G2/M arrest. This result differs from the G0/G1
arrest observed in SCC25 cells treated with 1 (ref. 27) but is consistent
with other work showing that 3 alone inhibits cell proliferation at the
G2/M checkpoint (ref. 28).

[0104]The combination of 1 and 3, but not 1 or 3 alone, induces mitotic
catastrophe. Assembly into microtubules is regulated by acetylation.
Tubulin deacetylation and destabilization is catalyzed by cytoplasmic
HDAC6, whose activity can be blocked by 3 (ref. 29). Results obtained in
cells treated with 1 and 3; such as the G2/M arrest, and the strong
induction of tubulin acetylation and hence microtubule stabilization,
suggest that the combination of 1 and 3 induces mitotic catastrophe in
SCC4 cells. Previous studies have shown that microtubule stabilizing
agents can induce mitotic catastrophe (ref. 30). SCC4 cells treated with
either 1 or 3 individually were morphologically similar (FIG. 12A) and
did not differ from control cells (not shown). In contrast, combined
treatment produced morphological changes, including variations in cell
size and shape, asymmetric cell divisions (FIG. 12A, asterisk), and the
formation of intercellular microtubular bridges (FIG. 12A, arrows)
reminiscent of telophase spindles (ref. 31). The formation of
intercellular microtubular structures was consistent with observations in
the light microscope of numerous intercellular bridges in cells treated
with both 1 and 3 (FIG. 12B, arrows), but not in other treatment groups
(not shown).

Experimental

[0105]General. MeCN, toluene and CH2Cl2 were distilled from
CaH2 under argon. THF and Et2O were distilled from sodium
metal/benzophenone ketyl under argon. All other commercial solvents and
reagents were used as received from the Aldrich Chemical Company, Fischer
Scientific Ltd., EMD Chemicals Inc., Strem or BDH. All glassware was
flame dried and allowed to cool under a stream of dry argon.

[0106]Silica gel (60 Å, 230-400 mesh) used in flash column
chromatography was obtained from Silicycle and was used as received.
Analytical thin-layer chromatography (TLC) was performed on pre-coated
silica gel plates (Ultra Pure Silica Gel Plates purchased from
Silicycle), visualized with a Spectroline UV254 lamp, and stained
with a 20% phosphomolybdic acid in ethanol solution, or a basic solution
of KMnO4. Solvent systems associated with Rf values and flash
column chromatography are reported as percent by volume values.

[0166](6S)-6-((1R,3R,7E,17β)-1,3-bis[tert-butyl(dimethyl)silyl-oxy]-9-
,10-secoestra-5,7-dien-17-yl)heptanoic acid (38) was prepared following
the procedure as for 35.

[0167](R)-5-((1R,3aS,7aR,E)-4-(2-((3R,5R)-3,5-dihydroxy cyclohexyl
idene)ethylidene)-7a-methyl-octahydro-1H-inden-1-yl)-N-hydroxyheptanamide
(39) was prepared from 38 following the same procedure as for 36.

[0168]It is to be understood that the invention is not limited in its
application to the details of construction and parts as described
hereinabove. The invention is capable of other embodiments and of being
practiced in various ways. It is also understood that the phraseology or
terminology used herein is for the purpose of description and not
limitation. Hence, although the present invention has been described
hereinabove by way of illustrative embodiments thereof, it can be
modified, without departing from the spirit, scope and nature of the
subject invention as defined in the appended claims.